This research focuses on oxidative modification of proteins. The resulting covalent modifications have been implicated in important physiologic and pathologic processes. Determination of the actual roles of oxidative modification in these processes requires the identification of specific proteins that are susceptible to modification and the mapping of the sites of modification in those proteins. During this year, emphasis continued on the development and improvement of methods for the detection of modified proteins and quantitation of those modifications. In particular, we have pursued mass spectrometric methods for the identification of oxidatively modified proteins. The sensitivity of this approach had previously allowed identification of some of the oxidatively modified proteins of interest, but for others it was not yet sufficient for confident identification. A 5- to 10-fold increase in sensitivity was accomplished by optimization of a post-column mixing system to eliminate suppression by trifluoroacetic acid and by eliminating matrix effects from sample buffers, especially by avoiding guanidine salts. Also, recovery of microgram quantities of proteins was improved by the same factor through development of an alkylation procedure in the organic solvent hexafluoroisopropanol. These improved techniques have allowed us to identify specific proteins that are oxidatively modified in vivo and, in some cases, the specific sites of modification. For example, with our collaborators, we showed that annexin II and thioredoxin peroxidase 2 are glutathionylated in HeLa cells exposed to oxidative stress. Having identified proteins that are sensitive to various oxidative modifications, we can now study the physiologic and pathologic effects of the modifications and consider rational intervention to modulate the extent of modification. - oxidative stress, metal-catalyzed oxidation, protein identification, protein carbonyl